Sulfated fucoglucuronomannan

ABSTRACT

An enzyme which decomposes a sulfated fucoglucuronomannan and is useful in the field of glycotechnology; a process for producing the enzyme; a fucoidan fraction reduced in the number of the kinds of molecules and useful as a reagent in glycotechnology; a sulfated fucoglucuronomannan oligosaccharide; and processes for producing the fraction and oligosaccharide.

TECHNICAL FIELD

The present invention relates to a sulfatedfucoglucuronomannan-degrading enzyme which is useful in a field ofglycotechnology and a method for producing the enzyme, as well as afucoidan fraction containing less molecular species and a sulfatedfucoglucuronomannan oligosaccharide which are useful as reagents forglycotechnology and methods for producing the same.

BACKGROUND ART

Brown algae contain a variety of sulfated polysaccharides. Thesesulfated polysaccharides are often generically called fucoidans orfucoidins. Their structures vary depending on the algae from which theyderive. For example, sulfated polysaccharides extracted from algae ofthe order Fucales, Kjellmaniella crassifolia Miyabe, Laminaria japonicaAreschoug, Cladosiphon okamuranus Tokida, Nemacystus decipiens(Suringar) Kuckuck and sporophyll of Undaria pinnatifida (Harvey)Suringar have structures different from each other. In general, asulfated polysaccharide fraction prepared from one alga contains amixture of several sulfated polysaccharide molecular species.

Sulfated polysaccharide molecular species of which the structures havebeen determined include sulfated fucans, sulfated fucoglucuronomannans,sulfated fucogalactans and sulfated glucuronofucans. Sulfatedpolysaccharides generally have some biological activities in many cases.For example, a sulfated fucan fraction has been reported to have astrong anticoagulant activity, and a sulfated fucoglucuronomannanfraction has been reported to have an apoptosis inducing activityagainst tumor cells. Therefore, attempts have been made in order todevelop a pharmaceutical using a sulfated polysaccharide.

If one intends to develop a pharmaceutical using a sulfatedpolysaccharide, it is necessary to determine its structure. Use of anenzyme that degrades the sulfated polysaccharide is very advantageousfor the determination of the structure. However, no enzyme that degradesa sulfated polysaccharide derived from a brown alga is commerciallyavailable. Furthermore, an enzyme that specifically degrades thesulfated polysaccharide is required in order to determine the structureof the sulfated polysaccharide. This is because sulfated polysaccharidesderived from brown algae vary depending on the species of the algae.

A sulfated polysaccharide mixture derived from an alga of the orderFucales has been reported to have an anticoagulant activity, an activityof inhibiting colonization by a chlamydia onto uterine epidermal cells,an activity of suppressing an allergic reaction, an activity ofsuppressing grafted organ rejection and the like. Structures offucoidans derived from algae of the order Fucales have been studied inorder to elucidate the relationship between the activities and thestructures. However, only average values for the structures have beenproposed based on physicochemical analyses.

Several kinds of sulfated polysaccharide molecular species are presentin a sulfated polysaccharide mixture fraction prepared from an alga ofthe order Fucales. Sulfated polysaccharides other than the molecularspecies that is responsible for the biological activity of interest aregenerally unnecessary. In some cases, such unnecessary molecular speciesmay induce ill effects.

It would be very useful for elucidating the relationship between thebiological activities and the structures if one could prepareoligosaccharides from a sulfated polysaccharide derived from an alga ofthe order Fucales with structural reproducibility. For example, anenzyme that degrades a sulfated fucoglucuronomannan contained in asulfated polysaccharide mixture fraction derived from a brown alga togenerate oligosaccharides is known (WO 96/34004). This enzyme acts wellon a sulfated fucoglucuronomannan derived from a brown alga of the orderLaminariales to generate sulfated fucoglucuronomannan oligosaccharides.However, it has almost no activity on a sulfated fucoglucuronomannanderived from a brown alga of the order Fucales.

For the reasons as described above, an enzyme that specifically degradesa molecular species contained in a sulfated polysaccharide mixturefraction derived from a brown alga of the order Fucales, a fucoidanfraction comprising more homogeneous molecular species, anoligosaccharide having an homogeneous structure produced by an enzymaticmeans, and methods for producing the same have been desired.

OBJECTS OF INVENTION

The main object of the present invention is to provide an enzyme thatefficiently degrades a sulfated fucoglucuronomannan derived from an algaof the order Fucales which is useful for glycotechnology and a methodfor producing the enzyme, as well as an oligosaccharide obtainable byallowing the enzyme to act on a sulfated fucoglucuronomannan and amethod for producing the same. Another object of the present inventionis to provide a fraction in which a sulfated fucoglucuronomannan isremoved from a sulfated polysaccharide mixture fraction derived from abrown alga and a method for producing the same.

SUMMARY OF INVENTION

As a result of intensive studies, the present inventors have found thata bacterial strain belonging to genus Fucophilus, Fucophilusfucoidanolyticus strain SI-1234, produces a novel sulfatedfucoglucuronomannan-degrading enzymes and a method for producing theenzyme. Furthermore, the present inventors have found that purity of afucoidan fraction can be increased by degrading and removing a sulfatedfucoglucuronomannan from a sulfated polysaccharide mixture fractionderived from a brown alga of the order Fucales utilizing the enzyme.Additionally, the present inventors have found that a novel sulfatedfucoglucuronomannan oligosaccharide having a homogeneous structure canbe produced from a sulfated polysaccharide mixture fraction derived froma brown alga of the order Fucales by utilizing the enzyme. Thus, thepresent invention has been completed.

The first aspect of the present invention relates to a sulfatedfucoglucuronomannan oligosaccharide of general formula (I) or (II), or asalt thereof:

wherein R is H or SO₃H.

The second aspect of the present invention relates to a sulfatedfucoglucuronomannan lyase having the following chemical and physicalproperties:

-   -   (I) acting on a sulfated fucoglucuronomannan derived from an        alga of the order Fucales and cleaving an α-D-mannosyl bond        eliminatively to generate an oligosaccharide having an        unsaturated glucuronate group;    -   (II) having an optimal pH of about 6.5 to 8.0; and

(III) having an optimal temperature of about 30 to 40° C.

The enzyme of the second aspect can be obtained according to a methodcomprising culturing a bacterium of the genus Fucophilus that is capableof producing the sulfated fucoglucuronomannan lyase and collecting saidenzyme from the culture.

The saccharide or a salt thereof of the first aspect can be preparedaccording to a method for producing the saccharide comprising allowingthe sulfated fucoglucuronomannan lyase of the second aspect to act on asulfated fucoglucuronomannan fraction derived from an alga of the orderFucales.

The third aspect of the present invention relates to a fucoidan fractionwhich is obtainable according to a method comprising removing a sulfatedfucoglucuronomannan converted into a smaller molecule by allowing thesulfated fucoglucuronomannan lyase of the second aspect to act on asulfated polysaccharide mixture fraction derived from a brown alga.

The fucoidan fraction of the third aspect can be prepared according to amethod for producing a fucoidan fraction comprising allowing thesulfated fucoglucuronomannan lyase of the second aspect to act on asulfated polysaccharide mixture fraction derived from a brown alga; andcollecting a fucoidan fraction.

The fourth aspect of the present invention relates to a reagent forglycotechnology which contains the sulfated fucoglucuronomannan lyase ofthe second aspect.

The fifth aspect of the present invention relates to a polysaccharidecomprising a sulfated fucoglucuronomannan of general formula (X):

wherein R is H or SO₃H; R₂ is H, SO₃H or general formula (XI); and n isan integer of 1 or more:

wherein R is H or SO₃H.

The sixth aspect of the present invention relates to a sulfatedfucoglucuronomannan having the following chemical and physicalproperties, or a salt thereof:

-   -   (1) containing fucose, mannose and glucuronic acid as        constituting saccharides; and    -   (2) converted into a smaller molecule by the action of the        sulfated fucoglucuronomannan lyase of the second aspect to        generate at least one compound selected from a compound of        general formula (I) or a compound of general formula (II):        wherein R is H or SO₃H,        wherein R is H or SO₃H.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1: a graph which illustrates the relationship between pH and therelative activity (%) of the sulfated fucoglucuronomannan lyaseaccording to the present invention.

FIG. 2: a graph which illustrates the relationship between temperature(° C.) and the relative activity (%) of the sulfated fucoglucuronomannanlyase according to the present invention.

FIG. 3: a figure which illustrates the ¹H-NMR spectrum of the sulfatedfucoglucuronomannan oligosaccharide 1-(1) according to the presentinvention.

FIG. 4: a figure which illustrates the mass spectrum of the sulfatedfucoglucuronomannan oligosaccharide 1-(1) according to the presentinvention.

FIG. 5: a figure which illustrates the ¹H-NMR spectrum of the sulfatedfucoglucuronomannan oligosaccharide 1-(2) according to the presentinvention.

FIG. 6: a figure which illustrates the mass spectrum of the sulfatedfucoglucuronomannan oligosaccharide 1-(2) according to the presentinvention.

FIG. 7: a figure which illustrates the ¹H-NMR spectrum of the sulfatedfucoglucuronomannan oligosaccharide 2-(2) according to the presentinvention.

FIG. 8: a figure which illustrates the mass spectrum of the sulfatedfucoglucuronomannan oligosaccharide 2-(2) according to the presentinvention.

FIG. 9: a figure which illustrates the ¹H-NMR spectrum of the sulfatedfucoglucuronomannan oligosaccharide 2-(3) according to the presentinvention.

FIG. 10: a figure which illustrates the mass spectrum of the sulfatedfucoglucuronomannan oligosaccharide 2-(3) according to the presentinvention.

FIG. 11: a figure which illustrates the ¹H-NMR spectrum of the sulfatedfucoglucuronomannan oligosaccharide 2-(4) according to the presentinvention.

FIG. 12: a figure which illustrates the mass spectrum of the sulfatedfucoglucuronomannan oligosaccharide 2-(4) according to the presentinvention.

FIG. 13: a figure which illustrates the ¹H-NMR spectrum of the sulfatedfucoglucuronomannan oligosaccharide 2-(5) according to the presentinvention.

FIG. 14: a figure which illustrates the mass spectrum of the sulfatedfucoglucuronomannan oligosaccharide 2-(5) according to the presentinvention.

DETAILED DESCRIPTION OF. THE INVENTION

The present invention will be explained in detail.

As used herein, a sulfated fucoglucuronomannan refers to a sulfatedpolysaccharide contained in a brown alga which contains fucose, mannoseand glucuronic acid as constituting saccharides. A substance having achemical structure represented by the above-mentioned general formula(I) and/or (II) is obtained by allowing the sulfated fucoglucuronomannanlyase of the present invention to act on the sulfatedfucoglucuronomannan. There is no specific limitation concerning theorigin of the sulfated fucoglucuronomannan. For example, sulfatedfucoglucuronomannans derived from brown algae of the order Fucales(e.g., Fucus vesiculosus, Ascophyllum nodosum) can be preferably used.As used herein, a sulfated fucoglucuronomannan fraction refers to afraction that contains a sulfated fucoglucuronomannan.

As used herein, a sulfated fucoglucuronomannan lyase refers to an enzymethat acts on a sulfated fucoglucuronomannan derived from a brown algaand cleaves an α-D-mannosyl bond between mannose and glucuronic acideliminatively to generate an oligosaccharide having an unsaturatedglucuronate group.

As used herein, a sugar compound refers to a sulfatedfucoglucuronomannan oligosaccharide, and includes a oligosaccharideobtainable by allowing the sulfated fucoglucuronomannan lyase of thepresent invention to act on a sulfated fucoglucuronomannan which hasmannose at the reducing end.

When a sulfated fucoglucuronomannan is produced according to the presentinvention, water-soluble components contained in a brown alga are firstextracted. In this case, it is preferable to carry out the extraction atpH 4-9 at a temperature of 100° C. or below in order to preventconversion of the sulfated fucoglucuronomannan into smaller molecules.Furthermore, amino acids or small molecule pigments in the extract canbe efficiently removed using ultrafiltration. Activated carbon treatmentis effective for the removal of hydrophobic substances.

Thus, a sulfated polysaccharide mixture fraction derived from a brownalga can be obtained. This fraction can be used as a sulfatedfucoglucuronomannan fraction, for example, as a substrate for thesulfated fucoglucuronomannan lyase of the present invention. A morehighly pure sulfated fucoglucuronomannan can be obtained by separatingthe sulfated fucoglucuronomannan fraction using an anion exchangecolumn. Either the sulfated polysaccharide mixture fraction or thesulfated fucoglucuronomannan purified using an anion exchange column maybe used as a substrate for determining an activity upon purification ofthe sulfated fucoglucuronomannan lyase of the present invention. Also,it may be used as a raw material for producing the fucoidan fractioncontaining less molecular species and the sulfated fucoglucuronomannanoligosaccharide of the present invention.

Any bacterium that produces the sulfated fucoglucuronomannan lyase ofthe present invention may be used for the production without limitation.For example, Fucophilus fucoidanolyticus strain SI-1234 can be used.

Fucophilus fucoidanolyticus strain SI-1234 is a bacterium newly obtainedby the present inventors by screening from a sea cucumber intestine. Itsbacteriological properties are as follows.

a. Morphological Properties:

-   -   (1) Coccus of 1.2 to 1.6 μm in diameter    -   (2) Spore: no    -   (3) Gram staining: negative        b. Physiological Properties:    -   (1) Growth temperature: 25° C.    -   (2) Attitude to oxygen: aerobic    -   (3) Catalase: positive    -   (4) Oxidase: negative    -   (5) Salt requirements:        -   Growth in 0% salt medium: negative        -   Growth in 1% salt medium: negative        -   Growth in seawater medium: positive    -   (6) Quinones: menaquinone 7    -   (7) GC content of intracellular DNA: 52%    -   (8) OF-test: not generating acid    -   (9) Colony color: not generating characteristic colony pigment    -   (10) Motility: negative    -   (11) Gliding: negative    -   (12) Flagellum: no

This strain is classified into Group 4 (Gram-negative aerobic bacilliand cocci) according to the basic classification as described inBergey's Manual of Determinative Bacteriology, Vol. 9 (1994). However,this strain is greatly different from bacteria belonging to Group 4 inthat it has menaquinone 7 in its electron transport chain and the GCcontent is 52%.

The nucleotide sequence of the DNA encoding 16S rRNA (16S rDNA) of thestrain was determined (SEQ ID NO:1) and compared its homologies to thoseof known bacteria. As a result, there was no known bacterium thatexhibits high homology over the whole 16S rDNA region (about 1,500bases). If the homology over the whole 16S rDNA sequence is 90% or less,two bacterial strains do not belong to the same genus. Accordingly, thepresent inventors concluded that this strain is a bacterium that doesnot belong to a known genus but belongs to a new genus, and designatedas Fucophilus fucoidanolyticus strain SI-1234. The sulfatedfucoglucuronomannan lyases of the present invention include a sulfatedfucoglucuronomannan lyase obtained from a bacterium that is determinedto belong to the same genus as Fucophilus fucoidanolyticus strainSI-1234 based on the nucleotide sequence of the 16S rDNA.

This strain is indicated as Fucophilus fucoidanolyticus strain SI-1234and deposited under Budapest Treaty at International Patent OrganismDepositary, National Institute of Advanced Science and Technology (AISTTsukuba Central 6, 1-1, Higashi 1-Chome, Tsukuba, Ibaraki 305-8566,Japan) on Aug. 18, 1999 (date of transmission: Mar. 7, 2001) underaccession number FERM BP-7495.

Any nutrient source can be added to a medium for culturing amicroorganism producing the sulfated fucoglucuronomannan lyase of thepresent invention as long as the microorganism utilizes it to producethe enzymes. For example, a sulfated fucoglucuronomannan, an alga suchas Fucus vesiculosus or Ascophyllum nodosum, alginic acid, laminaran,fucose, glucose, mannitol, glycerol, saccharose, maltose, starch and thelike can be utilized as carbon sources. Yeast extract, peptone, casaminoacid, corn steep liquor, meat extract, defatted soybean, ammoniumsulfate, ammonium chloride, urea, uric acid and the like are suitablenitrogen sources. In addition, a chloride, a phosphate or a sulfate ofsodium, potassium, magnesium, calcium, zinc or the like may be added.Generally, a microorganism obtained from seawater grows very well inseawater or commercially available artificial seawater.

The culture conditions are determined such that the productivity of thesulfated fucoglucuronomannan lyase of the present invention becomesmaximal depending on the microorganism used, the composition of themedium or the like. Generally, the maximal productivity of the sulfatedfucoglucuronomannan lyase of the present invention is achieved byculturing with aeration and stirring at a culture temperature of 15 to30° C. at medium pH of 5 to 9 for 5 to 72 hours. The sulfatedfucoglucuronomannan lyase of the present invention can be obtained fromcells and a culture supernatant separated from each other bycentrifugation after culturing.

A cell-free extract can be obtained by culturing Fucophilusfucoidanolyticus strain SI-1234 in an appropriate medium, collecting thecells, and disrupting the cells by a conventional means for celldisruption such as sonication. A purified enzyme preparation can be thenobtained from the extract by a conventional means for purification. Thesulfated fucoglucuronomannan lyase of the present invention in apurified form which is substantially free from other sulfatedfucose-containing polysaccharide-degrading enzymes can be obtained bypurification. For example, salting out, ion exchange columnchromatography, hydrophobic column chromatography or gel filtration maybe used for the purification.

Furthermore, a purification procedure similar to that for thepurification of the intracellular enzyme can be used to purify thesulfated fucoglucuronomannan lyase of the present invention from theculture supernatant which also contains the enzyme in large quantities.

The chemical and physical properties of the sulfated fucoglucuronomannanlyase of the present invention are as follows:

-   -   (I) acting on a sulfated fucoglucuronomannan and cleaving an        α-D-mannosyl bond eliminatively to generate an oligosaccharide        having an unsaturated glucuronate group;    -   (II) having an optimal pH of about 6.5 to 8.0 (FIG. 1, a graph        illustrating the relationship between the reaction pH and the        relative activity of the enzyme of the present invention, in        which the vertical axis represents the relative activity (%) and        the horizontal axis represents the pH);    -   (III) having an optimal temperature of about 30 to 40° C. (FIG.        2, a graph which illustrates the relationship between the        reaction temperature and the relative activity of the enzyme of        the present invention, in which the vertical axis represents the        relative activity (%) and the horizontal axis represents the        temperature (° C.)); and    -   (IV) having a molecular weight of about 500,000 to 600,000 as        determined by gel filtration.

The sulfated fucoglucuronomannan lyase of the present invention can beidentified by measuring an activity of degrading a sulfatedfucoglucuronomannan. A cell-free extract from a producer strain or anenzyme solution obtained after purification using various columnchromatographies may be used for the measurement.

Fucophilus fucoidanolyticus strain SI-1234 is a microorganism thatutilizes a sulfated fucoglucuronomannan. It produces the sulfatedfucoglucuronomannan lyase of the present invention inside and outsidethe cells for degrading the sulfated fucoglucuronomannan.

As used herein, a sulfated polysaccharide mixture fraction derived froma brown alga refers to a fraction that contains a fucoidan derived froma brown alga, i.e., a mixture of fucose-containing sulfatedpolysaccharides which is extracted from a brown alga.

According to the present invention, a fucoidan fraction containing lessmolecular species can be obtained by degrading a sulfatedfucoglucuronomannan contained in a sulfated polysaccharide mixturefraction with an enzyme and removing it.

The fucoidan fraction containing less molecular species may be obtainedby allowing the sulfated fucoglucuronomannan lyase to act on a sulfatedpolysaccharide mixture fraction extracted from a brown alga and removinga sulfated fucoglucuronomannan converted into smaller molecules, i.e.,sulfated fucoglucuronomannan oligosaccharides. For example,ultrafiltration, gel filtration, anion exchange column treatment or thelike may be used for the purification. Optionally, desalting,lyophilization or the like may be carried out.

For example, a sulfated polysaccharide mixture fraction derived from analga of the order Fucales contains several kinds of sulfatedpolysaccharides such as a sulfated fucoglucuronomannan in addition to asulfated fucan. A fucoidan fraction containing less molecular speciescan be prepared by degrading and removing the sulfatedfucoglucuronomannan using a sulfated fucoglucuronomannan lyase. Thefucoidan fraction containing less molecular species and a sulfatedfucoglucuronomannan oligosaccharide generated by degrading the sulfatedfucoglucuronomannan are useful as reagents for glycotechnology.

Upon preparation of the fucoidan fraction containing less molecularspecies of the present invention, a sulfated polysaccharide mixturefraction derived from a brown alga may be dissolved according to aconventional method. The sulfated polysaccharide mixture fraction may bedissolved in the solution at the maximal concentration. However, theconcentration may be usually selected taking its operationality, theamount of the sulfated fucoglucuronomannan lyase of the presentinvention to be used for the degradation and the like intoconsideration. The solvent for the sulfated polysaccharide mixturefraction may be selected from water, buffers and the like depending onthe objects. Usually, the pH of the solution is nearly neutral. Theenzymatic reaction is usually carried out at about −30° C. Optionally,the fucoidan fraction containing less molecular species may be furtherpurified using ion exchange resin treatment, ultrafiltration or thelike, or they may be desalted, sterilized or lyophilized.

A substance contained in the fucoidan fraction containing less molecularspecies of the present invention has a sulfate group and/or a carboxylgroup in its molecule. Such groups react with various bases to formsalts. Since the substance contained in the fucoidan fraction containingless molecular species of the present invention is stable in a form ofsalt, it is usually provided, for example, in a form of salt withsodium, potassium and/or calcium one can convert the salt into thesubstance contained in the fucoidan fraction containing less molecularspecies of the present invention in a free form by utilizing a cationexchange resin such as Dowex 50W. Optionally, the salts may be subjectedto conventional salt exchange for other various desirable salts.

A substance contained in the fucoidan fraction containing less molecularspecies of the present invention can be converted into apharmaceutically acceptable salt. Examples of the salts include saltswith alkaline metals such as sodium and potassium, salts with alkalineearth metals such as calcium and magnesium, as well as salts withammonium and zinc.

The sulfated fucoglucuronomannan oligosaccharide of the presentinvention can be prepared by allowing the sulfated fucoglucuronomannanlyase of the present invention to act on a sulfated fucoglucuronomannanor a sulfated fucoglucuronomannan-containing material. For example, apartially purified preparation of a sulfated fucoglucuronomannan, asulfated polysaccharide mixture fraction derived from a brown alga, anaqueous solvent extract of a brown alga, or a brown alga itself can bepreferably used as the sulfated fucoglucuronomannan-containing material

Upon preparation of the sulfated fucoglucuronomannan oligosaccharide ofthe present invention, a sulfated fucoglucuronomannan or a sulfatedfucoglucuronomannan-containing material may be dissolved according to aconventional method. The sulfated fucoglucuronomannan or the sulfatedfucoglucuronomannan-containing material may be dissolved in the solutionat the maximal concentration. However, the concentration may be usuallyselected taking its operationality and the amount of the sulfatedfucoglucuronomannan lyase of the present invention to be used for thereaction into consideration. The solvent for the sulfatedfucoglucuronomannan may be selected from water, buffers and the likedepending on the objects. Usually, the pH of the solution is nearlyneutral. The enzymatic reaction is usually carried out at about 30° C.The molecular weight of the sulfated fucoglucuronomannan oligosaccharidecan be controlled by adjusting the amount of the sulfatedfucoglucuronomannan lyase of the present invention used for thereaction, the composition of the reaction mixture, the reaction time orthe like. The sulfated fucoglucuronomannan oligosaccharide of thepresent invention having more homogeneous molecular weight can beprepared by fractionating the sulfated fucoglucuronomannanoligosaccharide of the present invention obtained as described aboveusing molecular weight fractionation or anion exchange column. Aconventional means for molecular weight fractionation such as gelfiltration or ultrafiltration may be used. Optionally, the smallermolecules may be subjected to further purification procedure using ionexchange resin treatment, activated carbon treatment or the like, orthey may be desalted, sterilized or lyophilized. The sulfatedfucoglucuronomannan oligosaccharide of the present invention having astructure so homogeneous that one can determine the structure by NMRanalysis as described below can be obtained by such a procedure.Examples of the sulfated fucoglucuronomannan oligosaccharides includecompounds represented by formulas (I) and (II) below. Although it is notintended to limit the present invention, at least one of the Rs ispreferably SO₃H in formulas (I) and (II).

wherein R is H or SO₃H,

wherein R is H or SO₃H.

The sulfated fucoglucuronomannan oligosaccharide of the presentinvention has a sulfate group and a carboxyl group in its molecule. Suchgroups react with various bases to form salts. Since the sulfatedfucoglucuronomannan oligosaccharide of the present invention is stablein a form of salt, it is usually provided, for example, in a form ofsalt with sodium, potassium and/or calcium. One can convert the saltinto the sulfated fucoglucuronomannan oligosaccharide of the presentinvention in a free form by utilizing a cation exchange resin such asDowex 50W. Optionally, the salts may be subjected to conventional saltexchange for other various desirable salts.

The sulfated fucoglucuronomannan oligosaccharide of the presentinvention can be converted into a pharmaceutically acceptable salt.Examples of the salts include salts with alkaline metals such as sodiumand potassium, salts with alkaline earth metals such as calcium andmagnesium, as well as salts with ammonium and zinc.

The sulfated fucoglucuronomannan lyase of the present invention convertsa sulfated fucoglucuronomannan into smaller molecules. So, it can beused for the structural analysis of the sulfated fucoglucuronomannan.The sulfated fucoglucuronomannan oligosaccharide of the presentinvention can be used as a reagent for glycotechnology. For example, a2-aminopyridine (PA)-labeled oligosaccharide prepared by subjecting theoligosaccharide to PA-labeling according to the method as described inJP-B 5-65108 can be used as a substrate for a fucofuranosidase or a5-sulfated fucofuranosidase. Thus, a substance very useful as a reagentfor glycotechnology can be provided.

The sulfated fucoglucuronomannan of the present invention is apolysaccharide having a structure in which fucose side chains areextended from mannose in a sugar chain composed of glucuronic acid andmannose alternately bound to each other. The polysaccharides are oftenpolymerized via a crosslinkage of a divalent cation or the like.Although it is not intended to limit the present invention, themolecular weights after cleaving the crosslinkages range from 5,000 to2,000,000, preferably from 10,000 to 1,000,000. Although it is notintended to limit the present invention, one represented by generalformula (X) exemplifies the sulfated fucoglucuronomannan. In generalformula (X), n is an integer of 1 or more, preferably 5 to 2000, morepreferably 10 to 1000.

wherein R is H or SO₃H; R₂ is H, SO₃H or general formula (XI); and n isan integer of 1 or more:

wherein R is H or SO₃H.

EXAMPLES

The following examples further illustrate the present invention indetail but are not to be construed to limit the scope thereof.

Referential Example 1 Preparation of Sulfated Polysaccharide MixtureFraction Derived from Fucus vesiculosus

Dried Fucus vesiculosus alga was ground. 1 kg of the ground alga wassuspended in 10 L of 80% ethanol. The suspension was stirred at 25° C.for 3 hours, filtered, and then washed to obtain a residue. The residuewas suspended in 30 L of 30 mM phosphate buffer (pH 6.5) containing 100mM sodium chloride. The suspension was treated at 95° C. for 2 hours andthen cooled to 30° C. 100 g of activated carbon, 3000 U of alginatelyase (Nagase Biochemicals) and 3.75 L of ethanol were added thereto.The mixture was stirred for 24 hours, and then centrifuged to obtain asupernatant. The supernatant was concentrated to 4 L using anultrafiltration device equipped with hollow fibers with exclusionmolecular weight of 100,000. The solvent was exchanged for 100 mM sodiumchloride. The solution was cooled to 5° C. The pH was adjusted to 2.0with 0.5 N hydrochloric acid. The formed precipitate was removed bycentrifugation to obtain a supernatant. The pH of the supernatant wasadjusted to 8.0 with 1 N sodium hydroxide. The solution was concentratedto 2 L using the above-mentioned ultrafiltration device and the solventwas exchanged for 20 mM sodium chloride. Insoluble substances wereremoved by centrifugation. The solution was lyophilized to obtain 80 gof a sulfated polysaccharide mixture fraction derived from Fucusvesiculosus.

Referential Example 2 Preparation of Sulfated Polysaccharide MixtureFraction Derived from Ascophyllum nodosum

100 g of a sulfated polysaccharide mixture fraction derived fromAscophyllum nodosum was obtained from 1 kg of commercially availableAscophyllum nodosum powder according to the method as described inReferential Example 1.

Referential Example 3 Preparation of Sulfated Polysaccharide MixtureFraction Derived from Kjellmaniella crassifolia Miyabe

Commercially available Kjellmaniella crassifolia Miyabe was disruptedusing a cutter mill (Masuko Sangyo) to prepare chips. 38 g of a sulfatedpolysaccharide mixture fraction derived from Kjellmaniella crassifoliaMiyabe was obtained from 1 kg of the chips according to the method asdescribed in Referential Example 1.

Referential Example 4 Preparation of Sulfated FucoglucuronomannanFraction Derived from Fucus vesiculosus

7 g of the sulfated polysaccharide mixture fraction derived from Fucusvesiculosus as described in Referential Example 1 was dissolved in 700ml of 20 mM imidazole-hydrochloride buffer (pH 6.0) containing 100 mMsodium chloride. The solution was loaded onto a 5-L DEAE-CellulofineA-800 equilibrated with the same buffer. The sample was run, the columnwas washed with 10 L of the same buffer, and elution was carried outwith a gradient of 100 to 1600 mM sodium chloride to collect fractions(500 ml). The total sugar content and the total uronic acid content ofeach fraction were measured according to the phenol-sulfuric acid methodand the carbazole-sulfuric acid method, respectively. A fraction elutedwith 200-700 mM sodium chloride was concentrated by ultrafiltration(exclusion molecular weight of 100,000), desalted and lyophilized toobtain 1.3 g of a sulfated fucoglucuronomannan fraction derived fromFucus vesiculosus.

Referential Example 5 Preparation of Sulfated FucoglucuronomannanFraction Derived from Ascophyllum nodosum

1.1 g of a sulfated fucoglucuronomannan fraction derived fromAscophyllum nodosum was obtained from 7 g of the sulfated polysaccharidemixture fraction derived from Ascophyllum nodosum as described inReferential Example 2 according to the method as described inReferential Example 4.

Referential Example 6 Preparation of Sulfated FucoglucuronomannanFraction Derived from Kjellmaniella crassifolia Miyabe

7 g of the sulfated polysaccharide mixture fraction derived fromKjellmaniella crassifolia Miyabe as described in Referential Example 3was dissolved in 700 ml of 20 mM imidazole-hydrochloride buffer (pH 8.0)containing 150 mM sodium chloride. The solution was loaded onto a 5-LDEAE-Cellulofine A-800 equilibrated with the same buffer. The sample wasrun, the column was washed with 10 L of the same buffer, and elution wascarried out with a gradient of 150 to 1950 mM sodium chloride to collectfractions (500 ml). The total sugar content and the total uronic acidcontent of each eluted fraction were measured according to thephenol-sulfuric acid method and the carbazole-sulfuric acid method,respectively. A fraction eluted with 350-490 mM sodium chloride wasconcentrated by ultrafiltration (exclusion molecular weight of 100,000),desalted and lyophilized to obtain 1.32 g of a sulfatedfucoglucuronomannan fraction derived from Kjellmaniella crassifoliaMiyabe.

Referential Example 7 Method for Measuring Sulfated FucoglucuronomannanLyase Activity

When the sulfated fucoglucuronomannan lyase of the present invention isallowed to act on a sulfated fucoglucuronomannan, an oligosaccharidehaving an unsaturated glucuronate group is generated, resulting inincreased absorbance at 232 nm. A sulfated fucoglucuronomannan lyaseactivity was measured utilizing it as follows. The sulfatedfucoglucuronomannan lyase of the present invention acts on sulfatedfucoglucuronomannans derived from Fucus vesiculosus and Ascophyllumnodosum. It also acts on a sulfated fucoglucuronomannan derived fromKjellmaniella crassifolia Miyabe. Then, a sulfated fucoglucuronomannanderived from Kjellmaniella crassifolia Miyabe was used as a substratefor the measurements of activities because it can be readily prepared.

Briefly, 50 μl of 2.5% solution of the sulfated fucoglucuronomannanfraction derived from Kjellmaniella crassifolia Miyabe as described inReferential Example 6, 50 μl of 100 mM phosphate buffer (pH 7.5), 10 μlof 4 M sodium chloride and 10 μl of the sulfated fucoglucuronomannanlyase of the present invention were mixed together. After reacting at37° C. for 3 hours, 105 μl of the reaction mixture was diluted with 2 mlof cold water. The absorbance at 232 nm was measured. As controls, areaction mixture obtained by a reaction in which the solvent used forthe enzyme solution was used in place of the sulfatedfucoglucuronomannan lyase of the present invention, and a reactionmixture obtained by a reaction in which water was used in place of thesulfated fucoglucuronomannan fraction were similarly analyzed.

One unit of a sulfated fucoglucuronomannan lyase activity is defined asan amount of an enzyme that generates 1 μmol of an unsaturatedglucuronate group in 1 minute in the above-mentioned reaction system.The activity of the enzyme was calculated according to the followingformula:Δ₂₃₂×2.105/5.5/180/0.01=U/ml

-   -   Δ₂₃₂: Increased absorbance at 232 nm;    -   2.105: Volume of sample subjected to absorbance measurement        (ml);    -   5.5: Molecular extinction coefficient (/mM) of unsaturated        glucuronate group;    -   180: Reaction time (minutes);    -   0.01: Volume of enzyme solution (ml).

The amount of protein was determined by measuring the absorbance at 280nm of the enzyme solution. The calculation was carried out assuming theabsorbance of a solution containing a protein at a concentration of 1mg/ml as 1.0.

Example 1 Preparation of Sulfated Fucoglucuronomannan Lyase

Fucophilus fucoidanolyticus strain SI-1234 was inoculated into 600 ml ofa medium consisting of artificial seawater (Jamarin Laboratory) (pH 8.0)containing the sulfated polysaccharide mixture fraction derived fromFucus vesiculosus as described in Referential Example 1 and peptone atconcentrations of 0.2% and 1%, respectively, which had been autoclavedat 120° C. for 20 minutes, and cultured at 24° C. for 72 hours toprepare a seed culture. A 30-L jar fermentor containing 20 L of a mediumconsisting of artificial seawater (Jamarin Laboratory) (pH 8.0)containing the sulfated polysaccharide mixture fraction derived fromFucus vesiculosus as described in Referential Example 1 and peptone atconcentrations of 0.2% and 1%, respectively, as well as an antifoamingagent (KM70, Shin-Etsu Chemical) was treated at 120° C. for 20 minutes.The seed culture was inoculated into the medium and cultured at 125 rpmat 24° C. for 48 hours. After cultivation, the culture was centrifugedto obtain cells and a supernatant.

The cells were suspended in 600 ml of 20 mM imidazole-hydrochloridebuffer (pH 7.0) containing 400 mM sodium chloride and 10 mM calciumchloride, sonicated and centrifuged to obtain a supernatant. Thesupernatant was adequately dialyzed against the same buffer andcentrifuged to obtain a supernatant as a crude enzyme solution of thesulfated fucoglucuronomannan lyase of the present invention.

Sulfated fucoglucuronomannan lyase activities contained in the culturesupernatant and the crude enzyme solution were measured. As a result, 1mU and 2 mU of the activities were detected in the culture supernatantcorresponding to 1 ml of the medium and the cell extract correspondingto 1 ml of the medium, respectively.

Example 2 Preparation of Sulfated Fucoglucuronomannan OligosaccharidesUsing Crude Enzyme Solution of Sulfated Fucoglucuronomannan Lyase, asWell as Purification and Structural Analyses Thereof (1).

(1) Preparation

The sulfated fucoglucuronomannan oligosaccharides of the presentinvention were prepared by allowing the crude enzyme solution asdescribed in Example 1 to act on the sulfated polysaccharide mixturefraction derived from Fucus vesiculosus as described in ReferentialExample 1. Briefly, 5 g of the sulfated polysaccharide fraction derivedfrom Fucus vesiculosus was dissolved in 500 ml of 25 mMimidazole-hydrochloride buffer (pH 7.0) containing 300 mM sodiumchloride and 50 mM calcium chloride. 50 ml of the crude enzyme solutionas described in Example 1 was then added thereto. The mixture wasreacted at 25° C. for 4 days. A supernatant obtained by centrifuging thereaction mixture was subjected to an ultrafiltration device equippedwith hollow fibers with exclusion molecular weight of 10,000 to collecta fraction of oligosaccharides having molecular weight of 10,000 orless. This fraction was designated as a sulfated fucoglucuronomannanenzymatic digestion product fraction 1.

(2). Purification

The sulfated fucoglucuronomannan enzymatic digestion product fraction 1obtained in Example 2-(1) was desalted using a desalting apparatus(Micro Acilyzer G3, Asahi Kasei). Imidazole and sodium chloride wereadded thereto at final concentrations of 10 mM and 10 mM, respectively.The resulting mixture was loaded onto a 1-L DEAE-Cellulofine A-800column equilibrated with 10 mm imidazole-hydrochloride buffer (pH 6.0)containing 10 mM sodium chloride. After washing with 2 L of the samebuffer, elution and collection were then carried out with a gradient of10 to 1200 mM sodium chloride. The absorbance at 232 nm was measured foreach fraction. The total sugar content and the total uronic acid contentof each fraction were measured according to the phenol-sulfuric acidmethod and the carbazole-sulfuric acid method, respectively. As aresult, the fractions of the latter half of washing and the fractionseluted with 360 mM sodium chloride formed peaks for which the absorbanceat 232 nm, the total sugar content and the total uronic acid contentwere proportional to each other. The fractions in each peak were pooledand the pools were designated as oligosaccharide fractions 1-(1) and1-(2), respectively.

Water was added to the oligosaccharide fraction 1-(1) to make theelectric conductivity equivalent to that of 10 mMimidazole-hydrochloride buffer (pH 6.0) containing 5 mM sodium chloride.The mixture was loaded onto a 30-ml DEAE-Cellulofine A-800 columnequilibrated with 10 mM imidazole-hydrochloride buffer (pH 6.0)containing 5 mm sodium chloride. The column was washed with 60 ml of thesame buffer. The flow-through fraction was collected, concentrated to2.8 ml using an evaporator, loaded onto a Cellulofine GCL-25 column(2×32 cm) equilibrated with 10% ethanol, eluted with 10% ethanol fordesalting and then dried. Thus, 1.6 mg of the sulfatedfucoglucuronomannan oligosaccharide 1-(1) of the present invention wasobtained.

Water was added to the oligosaccharide fraction 1-(2) to make theelectric conductivity equivalent to that of 10 mMimidazole-hydrochloride buffer (pH 6.0) containing 200 mM sodiumchloride. The mixture was loaded onto a 20-ml DEAE-Cellulofine A-800column equilibrated with 10 mM imidazole-hydrochloride buffer (pH 6.0)containing 200 mM sodium chloride. The column was washed with 40 ml ofthe same buffer. Elution was carried out with a gradient of 200-500 mMsodium chloride. A fraction eluted with 240-320 mM sodium chloride wascollected, concentrated to 1.0 ml using an evaporator, loaded onto aCellulofine GCL-25 column (2×32 cm) equilibrated with 10% ethanol,eluted with 10% ethanol for desalting and then dried. Thus, 6.4 mg ofthe sulfated fucoglucuronomannan oligosaccharide 1-(2) of the presentinvention was obtained.

(3) Structural Analyses

The sulfated fucoglucuronomannan oligosaccharides 1-(1) and 1-(2) of thepresent invention obtained in Example 2-(2) were subjected to analysesof saccharides at the reducing ends and saccharide compositions afterfluorescence labeling with 2-aminopyridine. As a result, the saccharideat the reducing end for each of the sulfated fucoglucuronomannanoligosaccharides 1-(1) and 1-(2) of the present invention was determinedto be mannose. Regarding the neutral sugar composition, eacholigosaccharide consisted of fucose and mannose. Next, determination ofthe sulfuric acid content (measured according to the turbidimetricmethod using barium chloride) and the uronic acid content (measuredaccording to the carbazole-sulfuric acid method), mass spectrometricanalysis using a mass spectrometer API-III, (Perkin-Elmer Sciex) and NMRanalysis using a nuclear magnetic resonance apparatus JNM-α500 (NipponDenshi) were carried out. Samples to be analyzed were subjected tostructural analyses after exchange for heavy water according to aconventional method. Bonds of constituting saccharides were analyzedusing the HMBC method, a method for ¹H-detection of heteronuclei. TheDQF-COSY method and the HOHAHA method were used for assignment in¹H-NMR.

Physical properties of the sulfatedfucoglucuronomannan-oligosaccharides-1-(1) and 1-(2) of the presentinvention are shown below.

(a) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 1-(1) of the Present Invention

The results for mass spectrometric analysis and assignment in NMRanalyses are shown below. The ¹H-NMR spectrum and mass spectrum areillustrated in FIGS. 3 and 4, respectively. In FIG. 3, the vertical axisrepresents the signal intensity and the horizontal axis represents thechemical shift value (ppm). In FIG. 4, the vertical axis represents therelative intensity and the horizontal axis represents the m/z value.

Molecular weight: 484

MS m/z 483.3 [M−H⁺]⁻

The results of ¹H-NMR analyses are shown in Table 1. TABLE 1 Chemicalshift value (ppm) ¹H-NMR Chemical shift value, multiplicity, couplingconstant F1-1 5.08, d, 4.0 F1-2 3.78, m F1-3 3.86, dd, 3.5, 10.0 F1-43.73, m F1-5 4.18, m F1-6 1.12, d, 7.0 M-1 5.29, d, 1.5 M-2 4.24, dd,1.5, 3.0 M-3 3.90, dd, 3.0, 9.0 M-4 3.78, m M-5 3.78, m M-6 3.78, mΔGA-1 5.02, d, 7.0 ΔGA-2 3.65, t, 7.0 ΔGA-3 4.18, m ΔGA-4 5.68, d, 3.5ΔGA-5 — ΔGA-6 —

-   Saccharide composition: L-fucose:D-mannose:unsaturated-   glucuronic acid=1:1:1-   Sulfate group: None

The numbers for peak assignment in ¹H-NMR are as indicated in formula(III) below:

(b) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 1-(2) of the Present Invention

The results for mass spectrometric analysis and assignment in NMRanalyses are shown below. The ¹H-NMR spectrum and mass spectrum areillustrated in FIGS. 5 and 6, respectively. In FIG. 5, the vertical axisrepresents the signal intensity and the horizontal axis represents thechemical shift value (ppm). In FIG. 6, the vertical axis represents therelative intensity and the horizontal axis represents the m/z value.

Molecular weight: 724

MS m/z 723.1 [M−H⁺]⁻, 766.7 [M+2Na⁺−3H⁺]⁻, 790.5 [M+3Na⁺−4H⁺]⁻, 360.9[M−2H⁺]²⁻, 372.0 [M+Na⁺−3H⁺]²⁻

The results of ¹H-NMR analyses are shown in Table 2. TABLE 2 Chemicalshift value (ppm) ¹H-NMR Chemical shift value, multiplicity, couplingconstant F1-1 5.30, d, 4.0 F1-2 4.42, dd, 4.0, 10.0 F1-3 4.52, dd, 3.0,10.0 F1-4 4.06, d, 3.0 F1-5 4.13, m F1-6 1.09, d, 6.5 M-1 5.31, d, 2.5M-2 4.25, m M-3 3.91, m M-4 3.85, t, 9.0 M-5 3.93, m M-6 4.10, dd, 6.0,11.0 4.22, m ΔGA-1 5.18, d, 7.0 ΔGA-2 3.60, t, 7.0 ΔGA-3 4.20, dd, 3.0,7.0 ΔGA-4 5.59, d, 3.0 ΔGA-5 — ΔGA-6 —

-   Saccharide composition: L-fucose:D-mannose:unsaturated-   glucuronic acid=1:1:1-   Sulfate group: 3 molecules

The numbers for peak assignment in ¹H-NMR are as indicated in formula(IV) below:

Example 3 Preparation of Sulfated Fucoglucuronomannan OligosaccharidesUsing Crude Enzyme Solution of Sulfated Fucoglucuronomannan Lyase, asWell as Purification and Structural Analyses Thereof (2)

(1) Preparation

The sulfated fucoglucuronomannan oligosaccharides of the presentinvention were prepared by allowing the crude enzyme solution asdescribed in Example 1 to act on the sulfated fucoglucuronomannanfraction derived from Fucus vesiculosus as described in ReferentialExample 4. Briefly, 0.61 g of the sulfated fucoglucuronomannan fractionderived from Fucus vesiculosus was dissolved in 60 ml of 20 mM phosphatebuffer (pH 7.0) containing 400 mM sodium chloride. 6 ml of the crudeenzyme solution as described in Example 1, which had been dialyzedagainst 20 mm imidazole-hydrochloride buffer (pH 7.0) containing 300 mMsodium chloride, 5 mM EDTA and 5 mM sodium azide, was then addedthereto. The mixture was reacted at 25° C. for 7 days. A supernatantobtained by centrifuging the reaction mixture was subjected toultrafiltration (exclusion molecular weight of 10,000) to collect afraction of oligosaccharides having molecular weight of 10,000 or less.This fraction was designated as a sulfated fucoglucuronomannan enzymaticdigestion product fraction 2.

(2) Purification

The sulfated fucoglucuronomannan enzymatic digestion product fraction 2obtained in Example 3-(1) was concentrated to 40 ml. The concentrate wasloaded onto a Cellulofine GCL-1000 (4×87 cm) equilibrated with 10%ethanol. Fractionation was carried out such that each fraction contained10 ml of the eluate. The 70th to 100th fractions were collected, anddesalted using a desalting apparatus (Micro Acilyzer G3, Asahi Kasei).Imidazole was added thereto at a final concentration of 10 mM. Theresulting mixture was loaded onto a 80-ml DEAE-Cellulofine A-800 columnequilibrated with 10 mm imidazole-hydrochloride buffer (pH 6.0). Afterwashing with 160 ml of the same buffer, elution and collection were thencarried out with a gradient of 0-800 mM sodium chloride. The absorbanceat 232 nm was measured for each fraction. The total sugar content andthe total uronic acid content of each fraction were measured accordingto the phenol-sulfuric acid method and the carbazole-sulfuric acidmethod, respectively. As a result, distinct five peaks for which theabsorbance at 232 nm, the total sugar content and the total uronic acidcontent were proportional to each other were detected. The fractions ineach peak were pooled and the pools were designated as oligosaccharidefractions 2-(1) to (5) according to the salt concentrations used for theelution (from low to high).

Each of the oligosaccharide fractions 2-(1) to (5) was concentrated to 4ml using an evaporator, loaded onto a Cellulofine GCL-25 column (2×32cm) equilibrated with 10% ethanol and eluted with 10% ethanol fordesalting, and then dried. Thus, 3.5, 3.9, 1.9, 1.7 and 0.9 mg of thesulfated fucoglucuronomannan oligosaccharides 2-(1) to (5) of thepresent invention were obtained, respectively.

(3) Structural Analyses

The sulfated fucoglucuronomannan oligosaccharides 2-(1) to (5) of thepresent invention obtained in Example 3-(2) were subjected to analysesof saccharides at the reducing ends and saccharide compositions afterfluorescence labeling with 2-aminopyridine. As a result, the saccharideat the reducing end for each of the sulfated fucoglucuronomannanoligosaccharides 2-(1) to (5) of the present invention was determined tobe mannose. Regarding the neutral sugar composition, eacholigosaccharide consisted of fucose and mannose. Next, determination ofthe sulfuric acid content (measured according to the turbidimetricmethod using barium chloride) and the uronic acid content (measuredaccording to the carbazole-sulfuric acid method), mass spectrometricanalysis using a mass spectrometer API-III (Perkin-Elmer Sciex) and NMRanalysis using a nuclear magnetic resonance apparatus JNM-α500 (NipponDenshi) were carried out. Samples to be analyzed were subjected tostructural analyses after exchange for heavy water according to aconventional method. Bonds of constituting saccharides were analyzedusing the HMBC method, a method for ¹H-detection of heteronuclei. TheDQF-COSY method and the HOHAHA method were used for assignment in¹H-NMR.

Physical properties of the sulfated fucoglucuronomannan oligosaccharides2-(1) to (5) of the present invention are shown below.

(a) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 2-(1) of the Present Invention

As a result of the above-mentioned analyses, it was demonstrated thatthis substance was identical to the sulfated fucoglucuronomannanoligosaccharide 1-(1) of the present invention.

(b) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 2-(2) of the Present Invention

The results for mass spectrometric analysis and assignment in NMRanalyses are shown below. The ¹H-NMR spectrum and mass spectrum areillustrated in FIGS. 7 and 8, respectively. In FIG. 7, the vertical axisrepresents the signal intensity and the horizontal axis represents thechemical shift value (ppm). In FIG. 8, the vertical axis represents therelative intensity and the horizontal axis represents the m/z value.

Molecular weight: 564

MS m/z 563.0 [M−H⁺]⁻, 585.0 [M+Na⁺−2H⁺]⁻ 281.0 [M−2H⁺]²⁻

The results of ¹H-NMR analyses are shown in Table 3. TABLE 3 Chemicalshift value (ppm) ¹H-NMR Chemical shift value, multiplicity, couplingconstant F1-1 5.05, d, 4.0 F1-2 3.75, m F1-3 3.82, dd, 3.0, 11.0 F1-43.71, m F1-5 4.15, m F1-6 1.10, d, 6.0 M-1 5.27, s M-2 4.22, m M-3 3.88,dd, 2.5, 10.0 M-4 3.78, t, 10.0 M-5 3.95, m M-6 4.15, m 4.22, m ΔGA-14.99, d, 6.5 ΔGA-2 3.63, t, 6.5 ΔGA-3 4.15, m ΔGA-4 5.66, d, 3.5 ΔGA-5 —ΔGA-6 —

-   Saccharide composition: L-fucose:D-mannose:unsaturated-   glucuronic acid=1:1:1-   Sulfate group: 1 molecule

The numbers for peak assignment in ¹H-NMR are as indicated in formula(V) below:

(c) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 2-(3) of the Present Invention

The results for mass spectrometric analysis and assignment in NMRanalyses are shown below. The ¹H-NMR spectrum and mass spectrum areillustrated in FIGS. 9 and 10, respectively. In FIG. 9, the verticalaxis represents the signal intensity and the horizontal axis representsthe chemical shift value (ppm). In FIG. 10, the vertical axis representsthe relative intensity and the horizontal axis represents the m/z value.

Molecular weight: 644

MS m/z 321.3 [M−2H+]²⁻

The results of ¹H-NMR analyses are shown in Table 4. TABLE 4 Chemicalshift value (ppm) ¹H-NMR Chemical shift value, multiplicity, couplingconstant F1-1 5.25, d, 3.5 F1-2 4.32, dd, 3.5, 10.5 F1-3 4.02, dd, 3.5,10.5 F1-4 4.55, d, 3.5 F1-5 4.39, q, 6.5 F1-6 1.16, d, 6.5 M-1 5.29, d,2.0 M-2 4.20, dd, 2.0, 2.5 M-3 3.86, dd, 2.5, 7.0 M-4 3.75, m M-5 3.75,m M-6 3.75, m ΔGA-1 5.45, d, 7.0 ΔGA-2 3.57, t, 7.0 ΔGA-3 4.27, dd, 3.5,7.0 ΔGA-4 5.62, d, 3.5 ΔGA-5 — ΔGA-6 —

-   Saccharide composition: L-fucose:D-mannose:unsaturated-   glucuronic acid=1:1:1-   Sulfate group: 2 molecules

The numbers for peak assignment in ¹H-NMR are as indicated in formula(VI) below:

(d) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 2-(4) of the Present Invention

The results for mass spectrometric analysis and assignment in NMRanalyses are shown below. The ¹H-NMR spectrum and mass spectrum areillustrated in FIGS. 11 and 12, respectively. In FIG. 11, the verticalaxis represents the signal intensity and the horizontal axis representsthe chemical shift value (ppm). In FIG. 12, the vertical axis representsthe relative intensity and the horizontal axis represents the m/z value.

Molecular weight: 870

MS m/z 456.1 [M+2Na−4H⁺]²⁻, 445.0 [M+Na⁺−3H⁺]²⁻

The results of ¹H-NMR analyses are shown in Tables 5 and 6. TABLE 5Chemical shift value (ppm) ¹H-NMR Chemical shift value, multiplicity,coupling constant F1-1 5.29, d, 4.0 F1-2 4.45, dd, 4.0, 10.5 F1-3 4.59,dd, 3.0, 10.5 F1-4 4.15, d, 3.0 F1-5 4.26, q, 6.0 F1-6 1.10, d, 6.0 F2-15.19, s F2-2 4.12, m F2-3 3.75, m F2-4 3.75, m F2-5 3.75, m F2-6 1.10,d, 6.0

TABLE 6 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, coupling constant M-1 5.30, d, 2.0 M-2 4.24, m M-3 3.91, mM-4 3.83, t, 9.5 M-5 3.91, m M-6 4.09, dd, 6.0, 11.0 4.18, m ΔGA-1 5.17,d, 7.0 ΔGA-2 3.60, t, 7.0 ΔGA-3 4.19, dd, 3.0, 7.0 ΔGA-4 5.58, d, 3.0ΔGA-5 — ΔGA-6 —

-   Saccharide composition: L-fucose:D-mannose:unsaturated-   glucuronic acid=2:1:1-   Sulfate group: 3 molecules

The numbers for peak assignment in ¹H-NMR are as indicated in formula(VII) below:

(e) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 2-(5) of the Present Invention

The results for mass spectrometric analysis and assignment in NMRanalyses are shown below. The ¹H-NMR spectrum and mass spectrum areillustrated in FIGS. 13 and 14, respectively. In FIG. 13, the verticalaxis represents the signal intensity and the horizontal axis representsthe chemical shift value (ppm). In FIG. 14, the vertical axis representsthe relative intensity and the horizontal axis represents the m/z value.

Molecular weight: 950

MS m/z 1037.0 [M+4Na−5H⁺]⁻, 506.9 [M+3Na−5H⁺]²⁻, 495.8 [M+2Na⁺−4H⁺]²⁻

The results of ¹H-NMR analyses are shown in Tables 7 and 8. TABLE 7Chemical shift value (ppm) ¹H-NMR Chemical shift value, multiplicity,coupling constant F1-1 5.30, d, 4.0 F1-2 4.47, dd, 4.0, 11.0 F1-3 4.61,m F1-4 4.15, d, 2.5 F1-5 4.28, m F1-6 1.11, d, 6.0 F2-1 5.18, s F2-24.12, m F2-3 3.93, m F2-4 3.93, m F2-5 4.52, dq, 1.5, 6.5 F2-6 1.30, d,6.5

TABLE 8 Chemical shift value (ppm) ¹H-NMR Chemical shift value,multiplicity, coupling constant M-1 5.32, d, 2.0 M-2 4.26, m M-3 3.93, mM-4 3.84, t, 9.5 M-5 3.93, m M-6 4.10, dd, 7.0, 11.5 4.20, m ΔGA-1 5.17,d, 7.0 ΔGA-2 3.63, t, 7.0 ΔGA-3 4.19, m ΔGA-4 5.59, d, 3.0 ΔGA-5 — ΔGA-6—

-   Saccharide composition: L-fucose:D-mannose:unsaturated-   glucuronic acid=2:1:1-   Sulfate group: 4 molecules

The numbers for peak assignment in ¹H-NMR are as indicated in formula(VIII) below:

Example 4 Preparation of Sulfated Fucoglucuronomannan OligosaccharidesUsing Recombinant Sulfated Fucoglucuronomannan Lyase, as well asPurification and Structural Analyses Thereof

(1) Preparation

The sulfated fucoglucuronomannan oligosaccharides of the presentinvention were prepared by allowing the recombinant sulfatedfucoglucuronomannan lyase, which was produced by transferring a gene forthe sulfated fucoglucuronomannan lyase as described in WO 99/11797 intoEscherichia coli, to act on the sulfated fucoglucuronomannan fraction asdescribed in Referential Example 4. Briefly, 0.4 g of the sulfatedfucoglucuronomannan fraction derived from Fucus vesiculosus wasdissolved in 40 ml of 20 mM imidazole-hydrochloride buffer (pH 7.0)containing 400 mM sodium chloride. 5 ml of a solution of the recombinantsulfated fucoglucuronomannan lyase (corresponding to 66 U) was thenadded thereto. The mixture was reacted at 25° C. for 25 hours. Thereaction mixture was loaded onto a Cellulofine GCL-1000 column (4×87 cm)equilibrated with 10% ethanol. Fractionation was carried out such thateach fraction contained 9.5 ml of the eluate. The 83rd to 120thfractions were pooled, and the pool was designated as a sulfatedfucoglucuronomannan enzymatic digestion product fraction 3.

(2) Purification

The sulfated fucoglucuronomannan enzymatic digestion product fraction 3obtained in Example 4-(1) was desalted using a desalting apparatus(Micro Acilyzer G3, Asahi Kasei). Imidazole was added thereto at final aconcentration of 10 mM. The resulting mixture was loaded onto a 20-mlDEAE-Cellulofine A-800 column equilibrated with 10 mMimidazole-hydrochloride buffer (pH 6.0). After washing with 40 ml of thesame buffer, elution was then carried out with a gradient of 0-800 mMsodium chloride. The absorbance at 232 nm was measured for eachfraction. The total sugar content and the total uronic acid content ofeach fraction were measured according to the phenol-sulfuric acid methodand the carbazole-sulfuric acid method, respectively. As a result, peaksfor which the absorbance at 232 nm, the total sugar content and thetotal uronic acid content were proportional to each other were detectedat portions eluted with 200 mM and 300 mM sodium chloride. The fractionsin each peak were pooled and the pools were designated asoligosaccharide fractions 3-(1) and 3-(2).

The oligosaccharide fraction 3-(1) was concentrated to 1 ml using anevaporator, loaded onto a Cellulofine GCL-25 column (1.2×30 cm)equilibrated with 10% ethanol and eluted with 10% ethanol for desalting,and then dried. Thus, 0.7 mg of the sulfated fucoglucuronomannanoligosaccharide 3-(1) of the present invention was obtained.

The oligosaccharide fraction 3-(2) was concentrated to 1.0 ml using anevaporator, loaded onto a Cellulofine GCL-25 column (1.2×30 cm)equilibrated with 10% ethanol, eluted with 10% ethanol for desalting,and then dried. Thus, 1.6 mg of the sulfated fucoglucuronomannanoligosaccharide 3-(2) of the present invention was obtained.

(3) Structural Analyses

The sulfated fucoglucuronomannan oligosaccharides 3-(1) and 3-(2) of thepresent invention obtained in Example 4-(2) were subjected to analysesof saccharides at the reducing ends and saccharide compositions afterfluorescence labeling with 2-aminopyridine. As a result, the saccharideat the reducing end for each of the sulfated fucoglucuronomannanoligosaccharides 3-(1) and 3-(2) of the present invention was determinedto be mannose. Regarding the neutral sugar composition, eacholigosaccharide consisted of fucose and mannose. Next, determination ofthe sulfuric acid content (measured according to the turbidimetricmethod using barium chloride) and the uronic acid content (measuredaccording to the carbazole-sulfuric acid method), mass spectrometricanalysis using a mass spectrometer API-III (Perkin-Elmer Sciex) and NMRanalysis using a nuclear magnetic resonance apparatus JNM-α500 (NipponDenshi) were carried out. Samples to be analyzed were subjected tostructural analyses after exchange for heavy water according to aconventional method. Bonds of constituting saccharides were analyzedusing the HMBC method, a method for ¹H-detection of heteronuclei. TheDQF-COSY method and the HOHAHA method were used for assignment in¹H-NMR.

Physical properties of the sulfated fucoglucuronomannan oligosaccharides3-(1) and 3-(2) of the present invention are shown below.

(a) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 3-(1) of the Present Invention

As a result of the above-mentioned analyses, it was demonstrated thatthis substance had the same structure as that of an oligosaccharidepreviously reported.

The structure is shown as formula (IX) below:

(b) Physical Properties of the Sulfated FucoglucuronomannanOligosaccharide 3-(2) of the Present Invention

As a result of the above-mentioned analyses, it was demonstrated thatthis substance was identical to the sulfated fucoglucuronomannanoligosaccharide 2-(3) of the present invention.

The results of Example 3 were compared with those of Example 4. Althougha 200-fold or more amount of the sulfated fucoglucuronomannan lyase wasused in Example 4, the efficiency of oligosaccharide generation on thebasis of the substrate weight was less than one fifth. Furthermore, sixkinds of oligosaccharides were obtained in Example 3, while only twokinds of oligosaccharides were obtained in Example 4. Thus, the sulfatedfucoglucuronomannan lyase of the present invention efficiently degradesa sulfated fucoglucuronomannan derived from Fucus vesiculosus whereasthe recombinant sulfated fucoglucuronomannan lyase used in Example 4cannot degrade the sulfated fucoglucuronomannan derived from Fucusvesiculosus so efficiently.

Example 5

Determination of Molecular Weight of the Sulfated FucoglucuronomannanLyase of the Present Invention

The crude enzyme solution of the sulfated fucoglucuronomannan lyase asdescribed in Example 1 was subjected to gel filtration using a SephacrylS-200 column (4.4×100 cm) equilibrated with 10 mMimidazole-hydrochloride buffer (pH 7.5) containing 100 mM sodiumchloride, 10 mM calcium chloride and 5 mM sodium azide, and fractionatedsuch that each fraction contained 13.5 ml of the eluate. The activity ofthe sulfated fucoglucuronomannan lyase of the present invention wasmeasured for each fraction according to the method as described inReferential Example 7. Thus, the molecular weight of the sulfatedfucoglucuronomannan lyase of the present invention was determined to beabout 500,000 to 600,000.

Example 6 Preparation of Fucoidan Fraction Containing Less MolecularSpecies

In order to prepare a fucoidan fraction containing less molecularspecies using the sulfated polysaccharide mixture fraction derived fromFucus vesiculosus as described in Referential Example 1, 500 ml of 50 mMphosphate buffer (pH 7.0), 50 ml of 4 M sodium chloride, 10 ml of thecrude enzyme solution of the sulfated fucoglucuronomannan lyase asdescribed in Example 1 and 5 g of the sulfated polysaccharide fractionderived from Fucus vesiculosus as described in Referential Example 1were mixed together, and the mixture was reacted at 25° C. for 5 days.Sulfated fucoglucuronomannan oligosaccharides resulted from thedegradation were removed by dialysis to prepare the fucoidan fractioncontaining less molecular species of the present invention in which asulfated fucoglucuronomannan was removed from the sulfatedpolysaccharide fraction.

Example 7 Structure of Site of Action for Sulfated FucoglucuronomannanLyase

The saccharide at the reducing end of each oligosaccharide generatedusing the sulfated fucoglucuronomannan lyase of the present inventionwas mannose. Unsaturated glucuronic acid was present at the nonreducingend. The absorbance at 232 nm was increased as the reaction proceeds.Therefore, it was assumed that this is an enzyme that cleaves a mannosylbond between mannose and glucuronic acid in a sulfatedfucoglucuronomannan eliminatively like the one as described in WO96/34004.

If it is the enzyme as assumed, there should be a polysaccharide havinga structure in which fucose side chains are extended from mannose in asugar chain composed of glucuronic acid and mannose alternately bound toeach other (a sulfated fucoglucuronomannan) in the fucoidan derived fromFucus vesiculosus. Oxalic acid treatment is a known method for obtaininga polysaccharide consisting of mannose and glucuronic acid(glucuronomannan) from a sulfated polysaccharide mixture derived from abrown alga (Carbohydrate Research, vol. 125, 283-290 (1984)). A fucoidanderived from Fucus vesiculosus was treated with reference to this methodto analyze its structure in order to confirm if a sulfatedfucoglucuronomannan was contained in Fucus vesiculosus as assumed.

Specifically, 1 g of the sulfated polysaccharide mixture fractionderived from Fucus vesiculosus prepared as described in ReferentialExample 1 was dissolved in 100 ml of water. 4.5 g of oxalic acid wasadded thereto. The pH was adjusted to 1.0 with 6 M sodium hydroxide. Themixture was treated at 100° C. for 5 hours. The pH was adjusted to 8with 6 M sodium hydroxide. Ethanol was added at final concentration of85% to a supernatant obtained by centrifugation. After allowing to standfor 2 hours, the mixture was centrifuged to obtain a precipitate. Theprecipitate was dissolved in 3000 ml of 10 mM imidazole-hydrochloridebuffer (pH 8.0). The solution was loaded onto 100-ml DEAE-Cellulofineequilibrated with 10 mM imidazole-hydrochloride buffer (pH 8.0)containing 30 mM sodium chloride. After washing with the same buffer,elution was carried out with a gradient of 30 mM to 500 mM sodiumchloride. Fractionation was carried out such that each fractioncontained 10 ml of the eluate. The neutral sugar content and the uronicacid content of each fraction were measured according to thephenol-sulfuric acid method and the carbazole-sulfuric acid method,respectively. The main component containing neutral sugar and uronicacid was eluted with 120-250 mM sodium chloride. The fraction wascollected, desalted using a electrodialysis device, lyophilized, andsubjected to NMR analysis after exchange for heavy water. As a result,it was confirmed that this substance had the same structure as that ofglucuronomannan as described in Carbohydrate Research, vol. 125, 283-290(1984).

Thus, it was shown that the unsaturated glucuronic acid contained in theoligosaccharide of the present invention was generated from glucuronicacid in the sulfated fucoglucuronomannan by the action of the sulfatedfucoglucuronomannan lyase of the present invention.

In addition, it was shown that the fucoidan derived from Fucusvesiculosus contained a novel sulfated fucoglucuronomannan whichcontained fucofuranose.

Industrial Applicability

The present invention provides a novel sulfated fucoglucuronomannanlyase which can be used for structural analysis of a sulfatedfucoglucuronomannan and reproducible production of a sulfatedfucoglucuronomannan oligosaccharide as well as a method for producingthe enzyme. The present invention further provides a sulfatedfucoglucuronomannan oligosaccharide and a fucoidan fraction containingless molecular species which can be produced using the enzyme and areuseful as reagents for glycotechnology as well as methods for producingthe same.

Sequence Listing Free Text

-   -   SEQ ID NO:1: 16S rDNA region of Fucophilus fucoidanolyticus        SI-1234

1. A sulfated fucoglucuronomannan oligosaccharide of general formula (I)or (II), or a salt thereof:

Wherein R is H or SO₃H,

Wherein R is H or SO₃H.
 2. A sulfated fucoglucuronomannan lyase havingthe following chemical and physical properties: (I) acting on a sulfatedfucoglucuronomannan derived from an alga of the order Fucales andcleaving an α-D-mannosyl bond eliminatively to generate anoligosaccharide having an unsaturated glucuronate group; (II) having anoptimal pH of about 6.5 to 8.0; and (III) having an optimal temperatureof about 30 to 40° C.
 3. A method for producing the sulfatedfucoglucuronomannan lyase defined by claim 2, the method comprising:culturing a bacterium of the genus Fucophilus that is capable ofproducing the sulfated fucoglucuronomannan lyase defined by claim 2; andcollecting said enzyme from the culture.
 4. A method for producing asulfated fucoglucuronomannan oligosaccharide of general formula (I) or(II), or a salt thereof, the method comprising allowing the sulfatedfucoglucuronomannan lyase defined by claim 2 to act on a sulfatedfucoglucuronomannan-containing fraction derived from an alga of theorder Fucales.

wherein R is H or SO₃H,

wherein R is H or SO₃H.
 5. A fucoidan fraction which is obtainableaccording to a method comprising removing a sulfated fucoglucuronomannanconverted into a smaller molecule by allowing the sulfatedfucoglucuronomannan lyase defined by claim 2 to act on a sulfatedpolysaccharide mixture fraction derived from a brown alga.
 6. A methodfor producing a fucoidan fraction, the method comprising: allowing thesulfated fucoglucuronomannan lyase defined by claim 2 to act on asulfated polysaccharide mixture fraction derived from a brown alga; andcollecting a fucoidan fraction.
 7. A reagent for glycotechnology whichcontains the sulfated fucoglucuronomannan lyase defined by claim
 2. 8. Asulfated fucoglucuronomannan having the following chemical and physicalproperties, or a salt thereof: (1) containing fucose, mannose andglucuronic acid as constituting saccharides; and (2) converted into asmaller molecule by the action of the sulfated fucoglucuronomannan lyasedefined by claim 2 to generate at least one compound selected from acompound of general formula (I) or a compound of general formula (II):

wherein R is H or SO₃H,

wherein R is H or SO₃H.